Explore the vast range of titles available.

Synthetic hydrogels are widely used as biomimetic in vitro model systems to understand how cells respond to complex microenvironments. The mechanical properties of hydrogels are deterministic for many cellular behaviors, including cell migration, spreading, and differentiation. However, it remains a major challenge to engineer hydrogels that recapture the dynamic mechanical properties of native extracellular matrices. Here, we provide a new hydrogel platform with spatiotemporally tunable mechanical properties to assay and define cellular behaviors under light. The change in the mechanical properties of the hydrogel is effected by a photo-induced switch of the cross-linker fluorescent protein, Dronpa145N, between the tetrameric and monomeric states, which causes minimal changes to the chemical properties of the hydrogel. The mechanical properties can be rapidly and reversibly tuned for multiple cycles using visible light, as confirmed by rheological measurements and atomic force microscopybased nano-indentation. We further demonstrated real-time and reversible modulation of cell migration behaviors on the hydrogels through photo-induced stiffness switching, with minimal invasion to the cultured cells. Hydrogels with a programmable mechanical history and a spatially defined mechanical hierarchy might serve as an ideal model system to better understand complex cellular functions.

•We explain mechanisms of light-induced conformational change of photoactivatable proteins.•We describe strategies and studies of using photoactivatable proteins to control intracellular signaling pathways.•We highlight the advantages of using light to control intracellular signaling pathways with superior spatial and temporal resolution.•We discuss precautions to be used in designing experimental schemes of optogenetic control of cell signaling. Cells employ a plethora of signaling pathways to make their life-and-death decisions. Extensive genetic, biochemical, and physiological studies have led to the accumulation of knowledge about signaling components and their interactions within signaling networks. These conventional approaches, although useful, lack the ability to control the spatial and temporal aspects of signaling processes. The recently emerged optogenetic tools open exciting opportunities by enabling signaling regulation with superior temporal and spatial resolution, easy delivery, rapid reversibility, fewer off-target side effects, and the ability to dissect complex signaling networks. Here we review recent achievements in using light to control intracellular signaling pathways and discuss future prospects for the field, including integration of new genetic approaches into optogenetics.

Purpose We developed a bimolecular fluorescence complementation (BiFC) strategy using Dronpa, a new fluorescent protein with reversible photoswitching activity and fast responsibility to light, to monitor protein–protein interactions in cells. Procedures Dronpa was split at residue Glu164 in order to generate two Dronpa fragments [Dronpa N-terminal: DN (Met1–Glu164), Dronpa C-terminal: DC (Gly165–Lys224)]. DN or DC was separately fused with C terminus of hHus1 or N terminus of hRad1. Flexible linker [(GGGGS)×2] was introduced to enhance Dronpa complementation by hHus1–hRad1 interaction. Furthermore, we developed expression vectors to visualize the interaction between hMYH and hHus1. Gene fragments corresponding to the coding regions of hMYH and hHus1 were N-terminally or C-terminally fused with DN and DC coding region. Results Complemented Dronpa fluorescence was only observed in HEK293 cells cotransfected with hHus1–LDN and DCL–hRad1 expression vectors, but not with hHus1–LDN or DCL–hRad1 expression vector alone. Western blot analysis of immunoprecipitated samples using anti-c-myc or anti-flag showed that DN-fused hHus1 interacted with DC-fused hRad1. Complemented Dronpa fluorescence was also observed in cells cotransfected with hMYH–LDN and DCL–hHus1 expression vectors or hMYH–LDN and hHus1–LDC expression vectors. Furthermore, complemented Dronpa, induced by the interaction between hMYH–LDN and DCL–hHus1, showed almost identical photoswitching activity as that of native Dronpa. Conclusion These results demonstrate that BiFC using Dronpa can be successfully used to investigate protein–protein interaction in live cells. Furthermore, the fact that complemented Dronpa has a reversible photoswitching activity suggests that it can be used as a tool for tracking protein–protein interaction.